International Journal of Organ Transplantation Medicine Comparison of Differentiation of Induced Pluripotent Stem Cells and Bone-marrow Mesenchymal Stem Cells to Osteoblast: Osteogenesis versus Pluripotency

Background: Derivation of induced pluripotent stem cells (iPSCs) from various adult somatic cells through over-expression of pluripotent genes could allow for the unlimited autologous supply in regenerative medicine. On the other hand the generation of various progenitors from bone-marrow mesenchymal stem cells (MSCs) is justly well established.


INTRODUCTION
T wo types of stem cells are currently recognized: adult stem cells and embryonic stem cells (ESCs). Adult stem cells are harvested from different tissue sources and variously called multipotent mesenchymal stromal cells or mesenchymal stem cells [1][2][3][4][5]. Mesenchymal stem cells (MSCs) could differentiate into osteoblast, chondroblast, cardiomyocyte, or even cells of non-mesodermal derivation including hepatocytes and neurons [6]. Although bone-marrow MSCs are originally isolated from bone marrow, similar pop-ulations have been reported in other tissues such as adipose and umbilical cord blood tissue. Adult stem cells have limitations in their application because they cannot be propagated indefinitely in culture; number of these cells also decreases with aging. There is evidence that these cells may exhibit reduced proliferation and differentiation with aging [13][14][15][16][17]. ESCs are considered to be pluripotent stem cells and are derived from the inner cells mass. These cells are capable of differentiation into any cell types. In contrast to adult stem cells, ESCs can be cultured indefinitely while maintaining their pluripotency [18][19][20]. Because of ethical concerns association with the application of ESCs in regenerative medicine, there is paucity of information regarding their potential applications for tissue regeneration.
On the other hand, Yamanaka and Takahashi managed to reprogram the somatic cells to pluripotent ESC-like cells by over-expression of transcription factors oct4, sox-2, klf-4, c-myc, lin28 and nanog [21]. Stem cells obtained from this method is named "iPSCs." They closely resemble ESCs because they restore a genome associated with a pluripotent marker. There are reports of attempts to generate osteoblast and chondroblast progenitors from ESCs and iPSCs [22].
In this study, we compared differentiation of iPSCs and bone-marrow MSCs into osteoblast using a monolayer approach. Osteoblast markers found in our in vitro samples were carefully analyzed. We also attempted to correlate expression of pluripotency markers oct4, c-myc, sox-2, nanog, klf4 and lin28 in iPSCs and MSCs before differentiation into osteoblast.
Melting curve analyses and PCR product sequencing were performed to verify primer specificities. RT-PCR was repeated at least three times using the following conditions. Each of the reaction mixtures contained 10 μL of SYBR Green master mix (Applied Biosystems), 5 pM each of forward and reverse primers and 5 μL of 100 times diluted cDNA.
To synthesize cDNA, 1 μg of total RNA was used. The relative expression levels of each gene was determined with the 2−ΔΔCt method. The primer sequences used for qPCR are mentioned in Table 1.

In Vitro Osteogenic Assay
We seeded and expanded 5-10×10 4 iPSCs and also MSCs per 6-well plates until nearly confluent. BMSC medium was then supplemented with dexamethasone + Ascorbic acid + β-glycerophosphate (mineralization medium) that was changed two or three times per week for 4-6 weeks, when signs of mineralization were visible under bright-field microscopy.  [21]. Each test was repeated three times.

RESULTS
The results of alizarin red staining showed that the mineralization process, where a reddish purple mass was observed in some areas of culture, indicated a positive trend of osteogenesis in human bone-marrow MSCs. The mass was observed in both groups. Figures  1 and 2 show that the rate of osteogenesis in MSCs group increased significantly (p<0.05)  Figure 3 indicates the expression of osteocalcin and osteopontin in both groups. Expression of osteocalcin and osteopontin genes in MSCs group was significantly (p<0.05) higher than that in iP-SCs group. In the present study, we compared the expression of six genes in human MSCs and iPSCs. Our data showed a significantly (p<0.05) higher expression of oct4, c-myc and klf4 in iPSCs compared with that in another group (Fig 4). MSCs expressed significantly lower level of oct4 and c-myc pluripotent markers than that in iPSCs group. In contrast, expression of sox2, nanog, and lin28 was higher in MSCs compare with iPSCs group (p=NS) (Fig 4).

DISCUSSION
In this study the osteogenesis potential of bonemarrow MSCs and iPSCs reprogrammed from skin fibroblast were compared. We evaluated the expression of osteogenic markers, oseopontin and osteocalcin, and showed that the expression of osteoblast markers in MSCs was higher than that in iPSCs.
Our results showed that the expression of some pluripotent markers such oct4 and c-myc in iPSCs was significantly (p<0.05) higher than bone-marrow MSCs. On the other hand, our results showed the expression of some pluripotent markers such as sox-2, nanog and lin28 in bone-marrow MSCs were more than that in iPSCs (p=NS).
Oct4 and c-myc are widely accepted as markers for pluripotent stem cells such as ESCs and iPSCs [22]. The expression of oct4 has already been reported in several adult somatic cells [23]. Oct4 expression in differentiated cells challenges its role as a pure stem cell marker [24]. Tai and colleagues reported that oct4 expression in somatic cells is restricted to small populations of multipotent cells with high self-renewal capacity, namely the adult stem cells [23]. Recently, researchers succeeded to induce pluripotent stem cells from primary human fibroblasts by only oct4 and sox-2 reprogram factors [25]. In the present research, oct4, as the most important pluripotent factor, expressed in both MSCs and iPSCs. It seems that a higher expression of osteopontin and osteocalcin in MSCs compared with iPSCs may be attributed to other factors (besides pluripotency) required for differentiation of stem cells to osteoblast.
Ratajczak and colleagues suggested oct4 is an embryonic transcription factor that occurs at low concentrations in somatic cells [26]. Tsai and colleagues reported that over-expression of only oct4 and klf4 genes is sufficient to induce reprogramming without exogenous or endogenous c-myc [27]. We found that both cells studied expressed oct4 gene and that the expression of oct4 transcriptional factor was significantly higher in iPSCs than bone-mar-row MSCs. Izadpanah, et al, concluded that oct4 is not specific to pluripotent stem cells [28]. In keeping with their findings, our results also showed that oct4 was not specific to pluripotent stem cells. One possible explanation could be that MSCs have some properties of pluripotent stem cells while they are being considered adult stem cells. We previously showed that MSCs derived from adipocyte tissue endogenously express high levels of cmyc [29]. Therefore, these cells can be reprogrammed into iPSCs merely by oct4 expression. Our data showed that iPSCs expressed the main pluripotent stem cells markers, oct4 and c-myc more than MSCs. The other possible explanation could be based on Bhartia hypothesis who asserts that the true stem cells in adult body tissues are the very small embryonic-like stem cells (VSELs), whereas MSCs are actually progenitor stem cells that arise by asymmetric cell division of VSELs [30]. The higher expression of osteogenesis markers in MSCs differentiated to osteoblast in comparison with iPSCs can indicate that in addition to pluripotent genes, other factors might also play a role in the osteogenesis differentiation.